B.t. is a gram-positive soil bacterium that produces crystal proteins during sporulation which are specifically toxic to certain orders and species of insects. Many different strains of B.t. have been shown to produce insecticidal crystal proteins Compositions including B.t. strains which produce insecticidal proteins have been commercially available and used as environmentally acceptable insecticides because they are quite toxic to the specific target insect, but are harmless to plants and other non-targeted organisms.
A number of genes encoding crystal proteins have been cloned from several strains of B.t. A good overview is set forth in H. Hofte et al., Microbiol. Rev., 53, pp. 242-255 (1989). While this reference is not prior art with respect to the present invention, it provides a good overview of the genes and proteins obtained from B.T. and their uses, adopts a nomenclature and classification scheme for B.t. genes and proteins, and has an extensive bibliography.
The B.t. crystal protein is active in the insect only after ingestion. After ingestion by an insect, the alkaline pH and proteolytic enzymes in the mid-gut solubilize the crystal allowing the release of the toxic components. These toxic components disrupt the mid-gut cells causing the insect to cease feeding and, eventually, to die. In fact, B.t. has proven to be an effective and environmentally safe insecticide in dealing with various insect pests.
As noted by Hofte et al., the majority of insecticidal B.t. strains are active against insects of the order Lepidoptera, i.e., caterpillar insects. Other B.t. strains are insecticidally active against insects of the order Diptera, i.e., flies and mosquitoes, or against both lepidopteran and dipteran insects. In recent years, a few B.t. strains have been reported as producing crystal protein that is insecticidal to insects of the order Coleoptera, i.e., beetles.
The first isolation of a coleopterantoxic B.t. strain is reported by A. Krieg et al., in Z. angew. Ent., 96, pp. 500-508 (1983); see also A. Krieg et al., Anz. Schaedlingskde, Pflanzenschutz, Umweltschutz, 57, pp. 145-150 (1984) and U.S. Pat. No. 4,766,203, issued Aug. 23, 1988 of A. Krieg et al. The strain, designated B.t. var tenebrionis, is reported to be toxic to larvae of the coleopteran insects Agelastica alni (blue alder leaf beetle) and Leptinotarsa decemlineata (Colorado potato beetle). B.t. tenebrionis makes an insecticidal crystal protein reported to be about 65-70 kilodaltons (kDa) (U.S. Pat. No. 4,766,203; see also K. Bernhard, FEMS Microbiol. Lett. 33, pp. 261-265 (1986).
V. Sekar et al., Proc. Natl. Acad. Sci. U.S.A., 84, pp. 7036-7040 (1987), report the cloning and characterization of the gene for the coleopteran-toxic crystal protein of B.t. tenebrionis. The size of the protein, as deduced from the sequence of the gene, was 73 kDa, but the isolated protein contained primarily a 65 kDa component. Hofte et al., Nucleic Acids Research, 15, p. 7183 (1987), also report the DNA sequence for the cloned gene from B.t. tenebrionis, and the sequence of the gene is identical to that reported by Sekar et al. (1987).
McPherson et al., Bio,/Technology, 6, pp. 61-66 (1988), disclose the DNA sequence for the cloned insect control gene from B.t. tenebrionis, and the sequence is identical to that reported by Sekar et al. (1987). E. coli cells and Pseudomonas fluorescens cells harboring the cloned gene were found to be toxic to Colorado potato beetle larvae.
A coleopteran-toxic strain, designated B.t. var. san diego, is reported by C. Herrnstadt et al., Bio/Technology, 4, pp. 305-308 (1986), to produce a 64 kDa crystal protein that was toxic to various coleopteran insects: strong toxicity to Pyrrhalta luteola (elm leaf beetle); moderate toxicity to Anthonomus grandis (boll weevil), Leptinotarsa decemlineata (Colorado potato beetle), Otiorhynchus sulcatus (black vine weevil), Tenebrio molitor (yellow mealworm) and Haltica tombacina; and weak toxicity to Diabrotica undecimpunctata undecimpunctata (western spotted cucumber beetle).
The DNA sequence of the cloned coleopteran toxin gene of B.t. san diego is reported in C. Herrnstadt et al., Gene, 57, pp. 37-46 (1987); see U.S. Pat. No. 4,771,131, issued Sep. 13, 1988, of Herrnstadt et al. The sequence of the toxin gene of B.t. san diego is identical to that reported by Sekar et al. (1987) for the cloned coleopteran toxin gene of B.t. tenebrionis.
A. Krieg et al., J. Appl. Ent., 104, pp. 417-424 (1987), report that the strain B.t. san diego is identical to the B.t. tenebrionis strain, based on various diagnostic tests.
Another new B.t. strain, designated EG2158, is reported by W. P. Donovan et al., Mol. Gen. Genet., 214 pp. 365-372 (1988) to produce a 73 kDa crystal protein that is insecticidal to coleopteran insects. The toxin-encoding gene from B.t. strain EG2158 was cloned and sequenced, and its sequence is identical to that reported by Sekar et al. (1987) for the cloned B.t. tenebrionis coleopteran toxin gene. This coleopteran toxin gene is referred to as the cryIIIA gene by Hofte et al., Microbiol. Rev., 53, pp. 242-255 (1989).
U.S. Pat. No. 4,797,279, issued Jan. 10, 1989, of D. Karamata et al., discloses a hybrid B.t microorganism containing a plasmid from B.t. kurstaki with a lepidopteran toxin gene and a plasmid from B.t. tenebrionis with a coleopteran toxin gene. The hybrid B.t. produces crystal proteins characteristic of those made by B.t. kurstaki, as well as of B.t. tenebrionis.
European Patent Application Publication No. 0 303 379, published Feb. 15, 1989, of Mycogen Corporation, discloses a novel B.t. isolate identified as B.t. MT 104 which has insecticidal activity against both coleopteran and lepidopteran insects.
European Patent Application Publication No. 0 318 143, published May 31, 1989, of Lubrizol Genetics, Inc., discloses the cloning, characterization and selective expression of the intact partially modified gene from B.t. tenebrionis, and the transfer of the cloned gene into a host microorganism rendering the microorganism able to produce a protein having toxicity to coleopteran insects. Insect bioassay data for B.t. san diego reproduced from Herrnstadt et al., Bio/Technology, 4, pp. 305-308 (1986) discussed above, is summarized. The summary also includes data for B.t. tenebrionis, from another source; B.t. tenebrionis is reported to exhibit strong toxicity to Colorado potato beetle, moderate toxicity to western corn rootworm (Diabrotica virgifera virgifera) and weak toxicity to southern corn rootworm (Diabrotica undecimpunctata).
European Patent Application Publication No. 0 324 254, published Jul. 19, 1989, of Imperial Chemical Industries PLC, discloses a novel B.t. strain identified as A30 which has insecticidal activity against coleopteran insects.
European Patent Application Publication No. 0 328 383, published Aug. 16, 1989, of Mycogen Corporation, discloses a novel B.t. microorganism identified as B.t. PS40DI which has insecticidal activity against coleopteran insects.
European Patent Application Publication No. 0 330 342, published Aug. 30, 1989, of Mycogen Corporation, discloses a novel B.t. microorganism identified as B.t.PS86B1 which has insecticidal activity against coleopteran insects.
These latter four publications are not prior art with respect to the present invention.
B.t. tenebrionis, first reported by A. Krieg et al., was discovered in or near Darmstadt, Germany and it is believed that B.t. san diego, reported by Herrnstadt et al., was obtained from a location in or near San Diego, Calif. B.t. strain EG2158, reported by Donovan et al., was isolated from a sample of crop dust from Kansas. Thus, various B.t. strains that have been isolated from several widely separated geographical locations all contained an apparently identical coleopteran toxin gene, the cryIIIA gene.
There appear to be no reports in the literature of any new coleopteran toxin B.t. genes other than the unique B.t. gene first discovered in B.t. tenebrionis over seven years ago.
Moreover, even among the various B.t. strains that have been reported as having crystal proteins insecticidally active against coleopteran insects, none has been shown to have significant toxicity to the larvae and adults of the insect genus Diabrotica (corn rootworm), which includes the western corn rootworm (Diabrotica virgifera virgifera), the southern corn rootworm (Diabrotica undecimpunctata howardi) and the northern corn rootworm (Diabrotica barberi). The cryIIIC gene of the present invention expresses protein toxin having quantifiable insecticidal activity against the Diabrotica insects, among other coleopteran insects.